Heart failure is a serious medical problem: over 5 million people in the US affected. The cause for chronic heart failure is multifaceted and includes bioenergetic deficiency, Ca2+ overload, and oxidative stress. ADP is the key substrate for ATP formation. Moreover, ADP is a potent inhibitor for the opening of mitochondrial permeability transition pore (mPTP). Although the molecular identity of mPTP is still unsolved, 2 different concepts stand out. One indicates that adenine nucleotide translocase (ANT) is not required for mPTP, whereas the other depicts mPTP as a multi-protein complex, the ANT and the mitochondrial peptidyl-prolyl cis-trans isomerase known as cyclophilin-D (Cyp-D), are the key components. Interestingly, binding of ADP to ANT facilitates cardiolipin to stabilize respiratory chain supercomplexes so that the efficiency of ATP generation is enhanced. Oxidation of cardiolipin destabilizes these supercomplexes and has been linked to mitochondrial dysfunction associated with aging, diabetic cardiomyopathy, ischemia-reperfusion injury, and heart failure. Furthermore, ADP inhibits efflux of inorganic phosphate Pi, the key mitochondrial Ca2+ buffer that forms Ca2+-Pi complex. Finally, activation of the mitochondrial fission protein, DLP1 causes mitochondrial fission, ROS generation, and mPTP opening. Taken together, these results lead us to hypothesize that binding of ADP to ANT serves two fundamental roles in mitochondrial function: enhancing ATP generation efficiency by stabilizing cardiolipin-ETC complexes and inhibiting mPTP by decreasing ROS generation and DLP1 activation. Physiologically, ADP- mediated mPTP inhibition minimizes excessive mitochondrial ROS generation and Ca2+ release from mitochondria in order to optimize the effectiveness of excitation-contraction-metabolism (ECM) coupling. Pathologically, defects of this ADP regulatory mechanism lead to energetic failure, oxidative stress, and Ca2+ dysregulation that enhance cardiac vulnerability to injury. We propose 2 Specific Aims: Specific Aim 1: To determine the mechanisms for ADP inhibition of mPTP. Hypothesis: ADP stabilizes cardiolipin integrity and inhibits DLP1 activity in the mitochondria and thus protects against Cyp-D-independent mPTP opening. Moreover, ADP decreases ROS generation via mPTP inhibition, and thus minimizes feedback activation of mPTP by ROS. Specific Aim 2: To assess the role of ADP in cardiac protection. Hypothesis: The mPTP in hearts of diseased models exhibits increased sensitivity to Ca2+-induced opening due to its predisposition to the first hit stresses including oxidative stress, high DLP1 activity, and/or diminished Ca2+ buffering capacity. Maintenance of optimal matrix ADP levels alleviates this increased mPTP vulnerability. Disturbances in the interaction between metabolic signaling and Ca2+/redox/cell death signaling are fundamental in disease pathogenesis including cardiac diseases. The exploratory and high-risk ideas in this application, if validated, will break new ground in a wide spectrum of disease mechanisms and treatments.